Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

Hatano, Naoya; Hamada, Tatsuro

doi:10.1016/j.jprot.2012.05.048

Cited by 56 publications

(42 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The total protein concentration of the activated and 80 ϫ enriched fluid was measured by a BCA assay to be 22 ng/l. The simplicity of the fluid proteome is expected based on earlier studies (31,32), in which nepenthesin was found to be a major component. For the purposes of our study, we assume 22 ng/l represents the nepenthesin concentration in the 80 ϫ enriched fluid, recognizing that it is likely an overestimate.…”

Section: Resultsmentioning

confidence: 90%

“…Plants in this genus have long fascinated botanists interested in mechanisms of insect trapping and nutrient uptake (25,41), and more recently, they have captured the imagination of materials researchers developing new lubrication technology (42). The proteome of the unfractionated fluid is remarkably simple, containing three to nine major proteins in the unstimulated secretions (31,32). Two major components are nepenthesin I and II, where I is present at ϳ10-fold higher levels than II (43).…”

Section: Fig 6 Peptide Map For the N-terminal Portion Of The Xrcc4 mentioning

confidence: 99%

See 1 more Smart Citation

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Rey

Yang

Burns

et al. 2013

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous endjoining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners. Molecular & Cellular

show abstract

Section: Resultsmentioning

confidence: 90%

Section: Fig 6 Peptide Map For the N-terminal Portion Of The Xrcc4 mentioning

confidence: 99%

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Rey

Yang

Burns

et al. 2013

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…The role of bacteria in producing digestive enzymes together with those produced by the plant on attacking prey in the pitchers have been previously shown. This included various groups of hydrolytic and nonhydrolytic enzymes, e.g., peroxidase, β-glucanase, chitinase, and phosphatase [8,39]. Most of these are acidophilic enzymes, reflecting their optimal activity adapted to the conditions in pitcher fluids…”

Section: Discussionmentioning

confidence: 99%

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

et al. 2016

View full text Add to dashboard Cite

Pitchers are specialized digestive organs of carnivorous plants which evolved for trapping prey and represent a unique environment harboring hidden diversity of unexplored microbes forming transient hydrolytic microcosms. In this study, the diversity of bacterial communities in the pitcher fluids of seven local Nepenthes found in Thailand was assessed by tagged 16S ribosomal RNA (rRNA) gene amplicon sequencing on an Ion PGM™ platform. A total of 1,101,000 filtered sequences were obtained which were taxonomically classified into 20 phyla, 48 classes, 72 orders, 153 families, and 442 genera while the remainder (1.43 %) could not be assigned to any existing taxa. Proteobacteria represented the predominant members in closed pitchers and more diversified bacterial taxa particularly Bacteriodetes and Actinobacteria, showed increasing abundance in open pitchers containing insect bodies. Principal coordinate analysis revealed that distribution of bacterial taxa was not significantly related to the Nepenthes species but strongly correlated to the pH of the pitcher fluids (pH 1.7-6.7). Acidicella was a highly dominant bacterial genus in acidic pitcher fluids while Dyella and Mycobacterium were also common genera in most pitchers. A unique microbial community structure was found in Nepenthes ampullaria which could reflect their adaptation to digest leaf litter, in addition to insect prey. The work revealed the highly unexplored nature of bacterial microcosms in Nepenthes pitcher fluids and provides insights into their community structure in this unique ecological system.

show abstract

“…In addition to proteins identified in this study (Supplementary Tables 25-28), we obtained for phylogenetic analyses a number of previously published sequences of digestive fluid proteins 8,9,[72][73][74][75][76][77][78] (Supplementary Table 24). Although many protein and transcript sequences for possible digestive enzymes are available (for example, refs 17,79-84 ), we included only genes for which complete coding sequences were available and for which their presence in digestive fluid had been biochemically validated (Supplementary Table 24, last searched 20 January 2016).…”

Section: Go Enrichment Analysis Supplementarymentioning

confidence: 99%

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

et al. 2017

View full text Add to dashboard Cite

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.

show abstract

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

Cited by 56 publications

References 32 publications

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

Contact Info

Product

Resources

About